title: A novel chloroplast transformation vector compatible with the Gateway(®) recombination cloning technology.
abstract: To analyze the suitability of Gateway(®) vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway(®) destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3% total soluble protein (TSP). The presented results clearly demonstrate that the Gateway(®) recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway(®) recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.
authors.affiliation: Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Street 24, 3430, Tulln, Austria.
authors.forename: Johanna
authors.initials: J
authors.lastname: Gottschamel
authors.forename: Mohammad Tahir
authors.initials: MT
authors.lastname: Waheed
authors.forename: Jihong L
authors.initials: JL
authors.lastname: Clarke
authors.forename: Andreas G
authors.initials: AG
authors.lastname: Lössl
mesh_terms: D001665:Binding Sites; D036022:Capsid Proteins / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics*; D005822:Genetic Vectors; D018745:Genome, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D011995:Recombination, Genetic; D012270:Ribosomes / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D036022:Capsid Proteins; C104967:gene 10 protein, phage T7; D049452:Green Fluorescent Proteins